Yu et al. take advantage of the dual wavelength emission of terbium conjugated anti-BTK antibody to serve as the energy donor for two fluorescent energy acceptors with distinct excitation and emission spectra: G2-streptavidin-bound biotinylated tirabrutinib (detects free BTK) and D2-coupled second anti-BTK antibody that binds to a different BTK epitope (detects total BTK). Additionally, the use of a common fluorescence donor serves to normalize the detection of total and free BTK with respect to each other. The assay is characterized and quantified using full-length purified recombinant human BTK protein and PBMCs derived from healthy volunteers and patients with CLL. The authors demonstrate the assay's utility using cells derived from samples from patients with CLL and DLBCL.

BTK is expressed in B cells and myeloid cells and plays an essential role in the B-cell receptor (BCR) signaling pathway. Tirabrutinib (GS-4059/ONO-4059) is a second-generation, potent, selective, irreversible BTK inhibitor. It is currently in clinical development as a treatment for chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), and other lymphoid malignancies.